Hemorrhagic feline calicivirus, calicivirus and method for preventing calicivirus infection or disease

ABSTRACT

The present invention relates to a novel, isolated and purified hemorrhagic feline calicivirus FCV-DD1. The invention further embraces monovalent and multivalent vaccines containing the new FCV-DD1 strain. In addition, the invention encompasses methods of protecting felines against infection or preventing disease caused by feline calicivirus or in addition to other pathogens that comprises administering to the felines an immunologically effective amount of the monovalent and multivalent vaccines described herein. Also, the invention concerns methods for diagnosing or detecting the hemorrhagic feline calicivirus in a susceptible host, asymptomatic carrier and the like by detecting the presence of feline calicivirus FCV-DD1 or antibodies raised or produced against feline calicivirus FCV-DD1 antigen.

CROSS-REFERENCE TO RELATED U.S. APPLICATIONS

This nonprovisional application claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 60/609,480, filed on Sep.13, 2004. The prior application is incorporated herein by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING”

Not Applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a new isolated and purified strain ofvirulent, hemorrhagic feline calicivirus, vaccines produced therefromand the use of the vaccines to protect cats from calicivirus infectionor disease.

2. Description of the Related Art

All patents and publications cited in this specification are herebyincorporated by reference thereto in their entirety.

Feline calicivirus (FCV) often causes an acute crisis in multiple-catenvironments, particularly animal hospitals and, to a lesser extent,animal shelters. Typically, the FCV infection presents signs resemblingviral rhinotracheitis (FVR) by affecting the upper respiratory tractand, on occasion, producing joint pain and lameness. Additionally, theinfected cat will develop ulcers on the tongue and in the mouth region.Vesicles and erosions of the nasal passages, the hard palate and thetongue appear prevalent. Other symptoms of FCV disease include highfever, hair loss, skin ulcerations and edema (swelling) in the legs oraround the face. Depending on the virulence of the infecting stain, theFCV infection may become fatal. The primary method of transmission isthrough the oral route of infection but cats can also get the infectionfrom inhalation of infectious virus found in the saliva, feces or urineof infected cats.

The FCV infection can affect both domestic cats and some wild felinespecies. Since FVR and FCV comprise almost 90% of all feline respiratoryinfections, the availability of effective vaccines to prevent these twodiseases is of great significance. FCV is a single-stranded RNA viruscapable of mutating into new strains (J. N. Burroughs et al.,“Physio-chemical evidence for the re-classification of thecaliciviruses,” Journal Gen. Virol. 22:281-286 (1974)). Over sixty-fivefeline caliciviruses exist worldwide, which makes adequate protection byvaccination using a singly comprised vaccine largely incomplete anddifficult. Because the virus is capable of mutation, monovalent vaccinesbased on a single strain of FCV may not be sufficiently protectiveagainst other FCV stains (see, generally, N. C. Pedersen et al.,“Mechanisms for persistence of acute and chronic feline calicivirusinfections in the face of vaccination,” Veterinary Microbiol.47(1-2):141-156 (November 1995); A. Lauritzen et al., “Serologicalanalysis of feline calicivirus isolates from the United States andUnited Kingdom” Veterinary Microbiol. 56(1-2):55-63 (May 1997); T.Hohdatsu et al., “Totalizing feature of commercially available felinecalicivirus (FCV) vaccine immune sera against FCV field isolates,”Journal of Veterinary Medicine Sci., 61(3):299-301 (March 1999): A. D.Radford et al., “Comparison of serological and sequence-based methodsfor typing feline calicivirus isolates from vaccine failures,” Vet Rec.146(5):117-123 (Jan. 29, 2000)).

Another problem with FCV is that the virus is highly contagious,infected cats will continue to shed the virus for long periods of timeafter infection and recovered cats may remain lifelong carriers of theinfectious virus. Asymptomatic cats can even spread fatal disease toother healthy cats. Recent outbreaks have been reported in NorthernCalifornia and New England of two genetically diverse strains of highlyvirulent, hemorrhagic calicivirus that were particularly fatal to thefeline population in animal shelters, named FCV-Ari and FCV-Diva,respectively (N. C. Pedersen et al., “An isolated epizootic ofhemorrhagic-like fever in cats caused by a novel and highly virulentstrain of feline calicivirus,” Veterinary Microbiol. 73:281-300 (May2000); E. M. Schorr-Evans et al., An epizootic of highly virulent felinecalicivirus disease in a hospital setting in New England, “Journal ofFeline Medicine and Surgery 5:217-226 (2003)).

In the past, monovalent viral vaccines have been described and severalmanufactured to prevent feline diseases using a variety of antigens suchas the feline calicivirus F9 strain (U.S. Pat. No. 3,944,469 (J. L.Bittle et al.)), feline Chlamydia psittaci (U.S. Pat. Nos. 5,972,350 and5,242,686 (H.-J. Chu et al., feline leukemia virus (U.S. Pat. No.4,264,587 (N. C. Pedersen et al.)) and the like. Other calicivirusstrains such as the FCV-M8 and FCV-255 and feline rhinotracheitis virushave also been previously isolated and described for vaccine use (E. V.Davis et al., “Studies on the safety and efficacy of an intranasalfeline rhinotracheitis-calicivirus vaccine,” VM-SAC 71:1405-1410 (1976);D. E. Kahn et al., “Induction of immunity to feline caliciviraldisease.” Infect. Immun. 11:1003-1009 (1975): D. E. Kahn, “Felineviruses: pathogenesis of picornavirus infection in the cat,” Am. J. Vet.Research 32:521-531 (1971)). Further, U.S. Pat. No. 4,522,810 (N. C.Pedersen) describes a feline calicivirus vaccine that contains theFCV-2280 strain. U.S. Pat. No. 6,231,863 (B. Colan et al.) describesnucleotide sequences from the genome of the FCV-2280 strain, andvaccines using the nucleotide sequences of the capsid gene forpreventing feline calicivirus disease. U.S. Pat. No. 5,106,619 (G. P.Wiesehahn et al.) discloses the preparation of inactivated viralvaccines that include feline calicivirus among others. U.S. Pat. No.6,051,239 (L. Simpson et al.) describes oral vaccines that use amodified botulinum toxin in conjunction with antigens such as thecalicivirus.

More recently, a strain of FCV-Kaos was identified (K. F. Hurley et al.,“An Outbreak of virulent systemic feline calicivirus disease, J. Am. VetMed. Assoc. 224(2):241-249 (Jan. 15, 2004)) and, subsequently, bothFCV-Kaos and FCV-Ari strains were isolated (U.S. Patent Application No.20040180064 A1, Hemorrhagic feline calicivirus, pub. Sep. 16, 2004). Theisolated virulent systemic calicivirus (VS-FCV) strains, includingFCV-Kaos, FCV-Ari and FCV-Bellingham, have been described as comprisinga capsia protein including an amino acid residue selected from the groupconsisting of lysine (K) at amino acid position 448; glutamic acid (E)at amino acid position 452; lysine (K) at amino acid position 581; andaspartic acid (D) at amino acid position 581 (U.S. Patent ApplicationNo. 20040259225 A1, Virulent systemic feline calicivirus, pub. Dec. 23,2004).

Multivalent vaccines have been prepared or described to contain mixturesof many viral antigens such as Chlamydophila felis (formerly known asfeline Chlamydia psittaci) in combination with one or more pathogenscomprising feline leukemia virus, feline panleukopenia virus, felinecalicivirus, feline rhinotracheitis virus, feline acquiredimmunodeficiency virus, rabies, feline infectious peritonitis, Borreliaburgdorferi and the like (U.S. Pat. No. 6,004,563 (H.-J. Chu el al.)).Another mixture of Rickard isolate feline leukemia virus, felinerhinotracheitis virus, feline calicivirus and feline panleukemia virushas similarly been disclosed as a vaccine (U.S. Pat. No. 5,374,424 (W.H. Kelsey et al.)).

Unfortunately, none of the prior vaccines, that contain previously usedstrains of the feline calicivirus adequately protect the feline from theemerging hemorrhagic feline calicivirus strains. In the recenthemorrhagic feline calicivirus outbreaks, there were a significantnumber of deaths despite the fact that the cats had receivedvaccinations against the calicivirus.

As a consequence, there is a definite art-recognized need in theveterinary field to produce an efficacious, safe vaccine against thehighly virulent, hemorrhagic feline calicivirus infections. Anotherart-recognized need is to provide a broad-spectrum viral vaccine thatprotects cats against serious infection and disease caused by bothhemorrhagic and common PCV strains. The novel FCV strain of the presentinvention is able to satisfy those needs by uniquely and advantageouslyeliciting specific immune response against the virulent, hemorrhagicstrain of FCV to protect cats from acute and chronic viral disease. Incombination with common calicivirus strains, the new FCV strain of thisinvention is able to achieve excellent virus-neutralizing antibodytiters and make broad-spectrum immunization possible.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns a highly infectious, novel hemorrhagicFCV strain, designated FCV-DD1, which is useful as a vaccine strain. Afurther embodiment of the invention is drawn to new methods of using thevaccine to immunize cats against infection and disease caused by thehemorrhagic feline calicivirus. Also embraced by this invention aremethods for diagnosing or detecting the hemorrhagic feline calicivirusin a susceptible host, asymptomatic carrier and the like by detectingthe presence of feline calicivirus FCV-DD1 or antibodies raised orproduced against feline calicivirus FCV-DD1 antigen.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS Not ApplicableDETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a new,highly infectious strain of feline calicivirus (FCV) and veterinaryvaccines to protect cats from viral infection caused by the calicivirus.More specifically, the invention describes an isolated and purifiedhemorrhagic feline calicivirus “FCV-DD1” and includes the viral clonesderived ton the FCV-DD1 isolate. (Whenever the FCV-DD1 isolate ismentioned herein, it is understood that the viral clones may substitutefor the parent isolate in each instance.) Also described are vaccinescontaining an immunogenic amount of FCV-DD1 and methods of protectingfelines against infection or preventing disease caused by felinecalicivirus that comprises administering to the feline in need ofprotection an immunologically effective amount of the vaccine. Thevaccine may optionally contain one or more additional FCV isolates suchas, for example, FCV-255, FCV-2280, FCV-Diva, FCV-Kaos, FCV-Bellingham,FCV-F9, FCV-F4, FCV-M8, etc. Desirably, the vaccine will contain FCV-DD1together with FCV-255, FCV-2280 or both, and, more preferably, themixture of FCV-DD1 with FCV-255.

Also, the vaccine may optionally contain other antigens or pathogenssuch as Chlamydophilia felis (C. felis), feline leukemia virus (FeLV),feline panleukopenia virus (FPV), feline rhinotracheitis virus (FVR),feline immunodeficiency virus, rabies virus, feline infectiousperitonitis virus, Bartonella bacteria (e.g. typical cat scratchdisease), a combination thereof and the like. Preferably, the mixture ofantigens comprises PCV-DD1 in combination with C. felis, feline leukemiavirus, feline panleukopenia virus and feline rhinotracheitis virus or incombination with C. felis, feline panleukopenia virus and felinerhinotracheitis virus. A particularly preferred multivalent vaccinecomprises FCV-DD2, a non-hemorrhagic feline calicivirus such as FCV-255,feline rhinotracheitis virus and feline panleukopenia virus, with theoptional addition of feline leukemia virus and/or C. felis, or other FCVstrains.

Leading up-to the discovery of the new hemorrhagic feline calicivirusFCV-DD1 strain, a tissue culture sample of FCV-Ari was obtained from Dr.Neils Pedersen at the School of Veterinary Medicine, UC Davis,California (R C. Pedersen et al., “An isolated epizootic ofhemorrhagic-like fever in cats caused by a novel and highly virulentstrain of feline calicivirus,” Veterinary Microbiol. 73:281-300 (May2000)). Toe sample of FCV-Ari was frozen, thawed and used to infect atissue culture roller bottle of confluent Crandell Feline Kidney Cells(CRFK) (R. A. Crandell et al., “Development, characterization, and viralsusceptibility of a feline (Felis catus) renal cell line (CRFK).” InVitro 9:176-185 (1973)). Later, the roller bottle was frozen, thawed andthe culture fluid was aliquoted as working stock.

The initial FCV-Ari “working stock” was used to inoculate cats in orderto confirm that the “working stock” from the material received from Dr.Neils Pedersen contained hemorrhagic calicivirus. The cats inoculatedwith the FCV-Ari “working stock” elicited extreme clinical signs such ashigh temperatures, edema, dehydration, and death confirming that theFCV-Ari “working stock” contained hemorrhagic calicivirus.

The FCV-Ari “working stock” was then diluted to a titer of 10⁵ TCID₅₀per mL, frozen, thawed, and 0.2 μm filtered. The filtered FCV-Ari wasused for subsequent purification and isolation of the most virulentcalicivirus strain. The filtered FCV-Ari was clarified, serially dilutedand used to infect 24-well tissue culture plates confluent with CRFKcells. The well of the highest dilution, which contained a cytopathiceffect (CPE) on the CRFK cells was harvested, frozen, quick, thawed,serially diluted, and used to infect another 24-well tissue cultureplate confluent with CRFK cells. This procedure was repeated two timesfor a total of three rounds of purification and isolation.

A portion of the thus-purified FCV-Ari was formalin inactivated and usedto blend a killed, monovalent vaccine. This inactivated FCV-Ari vaccinewas injected into cats to measure the serological response tovaccination. Unexpectedly, the vaccine failed to inducevirus-neutralizing antibody titers even though antibodies against thevirus were induced as confirmed by ELISA. In addition, the purifiedFCV-Ari (live) was used to inoculate two groups of cats. These twogroups of cats exhibited no clinical signs characteristic of ahemorrhagic calicivirus infection such as high temperatures, edema,pyoderma, alopecia, etc. Because the killed vaccine did not induceneutralizing antibodies and the live strain purified from the “workingstock” of FCV-Ari did not cause hemorrhagic calicivirus infection in acontrolled challenge study, the isolated virus from the firstpurification of the sample of FCV-Ari was confirmed not to be thehemorrhagic isolate; further work and vaccine development of this strainceased. It was then presumed that the original virus sample of FCV-Aricontained two FCV strains or possibly more, at least one of which wasnot virulent as demonstrated by the isolated strain that was obtainedfrom the first purification.

In an attempt to isolate the virulent strain that caused hemorrhagicfeline calicivirus infection, the original tissue culture sample ofFCV-Ari was used for three rounds of purification and isolation. Inorder to accomplish the task, the FCV-Ari sample was incubated with theantisera generated from the original FCV-Ari (killed) vaccination. Thevirus was serially diluted and used to infect 24-well tissue cultureplates confluent with CRFK cells, and the wells of the highest dilutionwhich displayed a cytopathic effect (CPE) on the CRFK cells wereharvested. The harvested virus clones, were evaluated by standard serumvirus-neutralization assays. Each viral clone was incubated with theantisera generated from the first purification killed vaccine, or withthe antisera generated from challenge with the live “working stock.” Theresults from this serum neutralization assay showed, surprisingly, thatthere was more than one strain of calicivirus in the original sample andfarther confirmed that the strain isolated from the first round ofpurification was not the hemorrhagic strain. The viral clones that werenot selected and discarded were those that were neutralized by theantisera specific to the undesired product of the first purification.The virus clones selected for the subsequent rounds of purification werethose that were neutralized by antisera to the original virus from Dr.Pedersen yet were not neutralized by antisera specific to the undesiredproduct of the first purification. Virus clone selection by harvestingthe highest dilution causing CPE was repeated for a total of threerounds. The resulting virus isolate, designated FCV-DD1, was inoculatedinto cats and the cats exhibited typical hemorrhagic calicivirusclinical signs. By this process, the purified and isolated FCV-DD1strain was determined to be a true hemorrhagic feline calicivirusstrain, previously unknown in the veterinary field.

Consequently, the new, purified and isolated FCV-DD1 strain, vaccines ofFCV-DD1 and methods of using the calicivirus are included within thescope of the present invention. Inoculated cats are protected fromserious viral infection and disease caused by the calicivirus. The novelmethod protects cats in need of protection against viral infection byadministering to the cat an immunologically effective amount of avaccine according to the invention, such as, for example, the vaccinecontaining killed, modified live or attenuated FCV-DD1 or its clone. Thevaccines may farther contain, additional antigens to promote theimmunological protection of cats against multiple feline diseasesincluding, but not limited to, non-hemorrhagic calicivirus strains,e.g., FCV-255, FCV-2280, etc., other hemorrhagic calicivirus strains,e.g., FCV-Diva, FCV-Kaos, FCV-F9, etc. and other suitable antigens suchas feline viral rhinotracheitis, feline panleukopenia virus (felinedistemper), Chlamydophila felis (C. felis), etc. The additional antigensmay be given concurrently to the cat in a combination product orseparately in order to provide a broad spectrum of protection againstviral infections. Most preferably, the mixture contains FCV-DD1,FCV-255, C. felis, feline leukemia virus, feline panleukopenia virus andfeline rhinotracheitis virus or, alternatively, FCV-DD1, FCV-255, felinepanleukopenia virus and feline rhinotracheitis virus, killed virus, and,optionally, feline leukemia virus and/or C. felis or other FCV strains.To broaden the scope of protection conferred by the FCV-DD1 containingvaccine against infection or disease in complementary fashion, it ishelpful to have the multivalent vaccine contain two or more FCV strainsin which the additional FCV strain may include, but is not limited to,FCV-255, FCV-2280, FCV-Diva, FCV-Kaos, FCV-Bellingham, FCV-F9, FCV-F4,FCV-M8, etc.; and it is particularly beneficial to include at least oneor more non-hemorrhagic strain such as FCV-255, FCV-2280, etc. Whencertain FCV antigens such as FCV-F9 are employed, it is desirable tomake a modified live or attenuated vaccine to accommodate the virulenceof the virus. A preferred combination of antigens in a vaccine is one inwhich the additional feline calicivirus with FCV-DD1 comprises FCV-255,PCV-2280 or the combination of FCV-255 and FCV-2280, in conjunction withat least feline panleukopenia virus and feline rhinotracheitis virus.

The vaccines comprise, for example, the infectious viral strain as aninactivated (killed) virus, an attenuated virus, a modified live virus,etc, in combination with a nontoxic, physiologically acceptable carrieror diluent and other inert excipients, adjuvants or conventionalco-formulants that are tolerated by the feline species. The isolated andpurified FCV-DD1 strain or its viral clone can be used as a monovalentvaccine in which protection relies on its ability to provide protectionagainst infection by other serotypes through cross-neutralization.Repeated inoculation with the same serotype typically confers protectionagainst subsequent severe infection.

The new vaccines of this invention are not restricted, to any particulartype or method of preparation. The viral vaccines include, but are notlimited to, inactivated (killed) vaccines, modified live vaccines,attenuated vaccines, subunit vaccines, genetically engineered, vaccines,etc. These vaccines are prepared by standard methods known in the art.The most preferred vaccines for delivery of the new FCV-DD1 strain toinoculate cats against the virulent FCV infection and disease are theinactivated (killed) or modified live virus vaccines.

To prepare inactivated virus vaccines, for instance, virus propagationis done by methods known in the art or described herein. Virusinactivation is achieved by protocols generally known to those ofordinary skill in the art. Inactivated virus vaccines may be prepared bytreating the virus with inactivating agents such as formalin orhydrophobic solvents, acids, beta propiolactone, binary ethyleneimine,etc. Formalin is the most preferred inactivating agent. Inactivation isconducted in a manner understood in the art. For example, to achievechemical inactivation, a suitable virus sample or serum samplecontaining the virus is treated for a sufficient length of time with asufficient amount or concentration of inactivating agent at asufficiently high or low temperature or pH, depending on theinactivating agent, to inactivate the virus. The virus is consideredinactivated if it is unable to infect a cell susceptible to infection.

The preparation of subunit vaccines typically differs from thepreparation of a modified live vaccine or an inactivated vaccine. Priorto preparation of a subunit vaccine, the protective or antigeniccomponents of the vaccine must be identified. Such protective orantigenic components include, for example, the immunogenic proteins orcapsid proteins of the virus strain. These immunogenic components areidentified by methods known in the art. Once identified, the protectiveor antigenic portions of the virus (i.e., the subunit) are subsequentlypurified by standard procedures and/or cloned by standard recombinantDNA techniques (see, for example, Maniatis et al., “Molecular Cloning: Alaboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor,Mass., 1989). The subunit vaccine provides an advantage over othervaccines based on the live virus since the subunit, such as highlypurified subunits of the virus, is less toxic than the whole virus.

To prepare attenuated vaccines from virulent viral clones, the tissueculture adapted, live pathogenic FCV is first attenuated by methodsknown in the art, typically made by serial passage through cellcultures. Attenuation of pathogenic clones may also be made byintroducing point mutations, effecting gene deletions in the virusgenome.

An immunologically effective or immunogenic amount of the vaccine of thepresent invention is administered to a feline in need of protectionagainst viral infection, usually 8 to 10 weeks of age or older. Theimmunologically effective or immunogenic amount that inoculates the catagainst FCV infection and disease can be easily determined or readilytitrated by routine testing by those of ordinary skill in the veterinaryfield. An effective amount is one in which a sufficient immunologicalresponse to the vaccine is attained to protect the cat exposed to thevirulent feline virus. This immunological response for FCV is generallyshown through the ability of the vaccine to induce virus-neutralizingantibody titers. Preferably, the cat is protected to an extent in whichone to all of the adverse physiological symptoms or effects of the viraldisease state are significantly reduced, ameliorated or totallyprevented.

The vaccine is typically administered in a single dose or repeateddosages over time. Dosages range, for example, from about 0.25 mL toabout 3.5 mL, usually about 0.5 mL to about 2.5 mL, preferably fromabout 0.8 mL to about 1.2 mL, and most preferably, at about 1.0 mL,depending upon the concentration of the immunogenic component of thevaccine, but should not contain an amount of virus-based antigensufficient to result in an adverse reaction or physiological symptoms ofviral infection. Methods are well known in the art for determining ortitrating suitable dosages of active antigenic agent to find minimumeffective dosages based on the weight of the cat, concentration of theantigen and other typical factors. For optimal immunization, a healthycat is vaccinated with a dose of approximately 1 mL using aseptictechnique and then a second 1 mL dose is given in about two to fourweeks later. Annual re-vaccination with a single booster shot of thevaccine is useful to maintain good immunity against infection.

The vaccine can conveniently be administered intranasally, transdermally(i.e., applied on or at the skin surface for systemic absorption),parenterally, orally, etc., or a combination such as oronasal where partof the dose is given orally and part is given into the nostrils. Theparenteral route of administration includes, but is not limited to,intramuscularly, subcutaueously, intradermally (i.e., injected orotherwise placed under the skin), intravenously and the like. Theintramuscular, subcutaneous and oronasal routes of administration aremost preferred.

When administered as a liquid, the present vaccine may be prepared inthe conventional form of an aqueous solution, syrup, elixir, tinctureand the like. Such formulations are known in the art, and are typicallyprepared by dissolution or dispersion of the antigen and other additivesin the appropriate carrier or solvent systems for administration tocats. Suitable nontoxic, physiologically acceptable carriers or solventsinclude, but are not limited to, water, saline, ethylene glycol,glycerol, etc. The vaccine may also be lyophilized or otherwisefreeze-dried and then aseptically reconstituted or rehydrated using asuitable diluent shortly before use. Suitable diluents include, but arenot limited to, saline, Eagle's minimum essential media and the like.Typical additives or co-formulants are, for example, certified dyes,flavors, sweeteners and one or more antimicrobial preservatives such asthimerosal (sodium ethylmercurithiosaliclate), neomycin, polymyxin B,amphotericin B and the like. Such solutions may be stabilized, forexample, by addition of partially hydrolyxed gelatin, sorbitol or cellculture medium, and may be buffered by conventional methods usingreagents known in the art, such as sodium hydrogen phosphate, sodiumdihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogenphosphate, a mixture thereof, and the like.

Liquid formulations also may include suspensions and emulsions thatcontain suspending or emulsifying agents in combination with otherstandard co-formulants. These types of liquid formulations may beprepared by conventional methods. Suspensions, for example, may beprepared using a colloid mill. Emulsions, for example, may be prepared,using a homogenizer.

Parenteral formulations, designed for injection into body fluid systems,require proper isotonicity and pH buffering to the corresponding levelsof feline body fluids. Isotonicity can be appropriated adjusted withsodium chloride and other salts as necessary. At the time ofvaccination, the virus is thawed (if frozen) or reconstituted (iflyophilized) with a physiologically-acceptable carrier such as deionizedwater, saline, phosphate buffered saline, or the like. Suitablesolvents, such as propylene glycol, can be used to increase thesolubility of the ingredients in the formulation and the stability ofliquid preparations.

Further additives that may be employed in the present vaccine include,but are not limited to, dextrose, conventional antioxidants andconventional chelating agents, such as ethylene-diamine tetraacetic acid(EDTA). Other pharmaceutically acceptable adjuvants that may optionallysupplement the vaccine formulation include, but are not limited to,surfactants, polyanions, polycations, peptides, mineral oil emulsion,immunomodulators, a variety of combinations and the like. Furthernon-limiting examples of suitable adjuvants include squalane andsqualene (or other oils of animal origin);polyoxyethylene-polyoxypropylene block copolymers such as Pluronic®(L121, for example, commercially available from BASF Aktiengesellschaft,Ludwigsshafen, Germany); saponin; detergents such as Tween®-80(polysorbate 80, commercially available from Sigma Chemical Co., St.Louis, Mo.): Quil A (commercial name of a purified form of Quiliajasaponaria, available from Iscotec AB, Sweden and Superfos Biosector a/s,Vedbaek, Denmark); mineral oils such as Marcol® (a purified mixture ofliquid saturated hydrocarbons, commercially available from ExxonMobil,Fairfax, Va.); vegetable oils such as peanut oil;Corynebacterium-derived adjuvants such as Corynebacterium parvum;Propionibacterium-derived adjuvants such as Propionibacterium acne:Mycobacterium bovis (Bacille Calmette-Guerin, or BCG); interleukins suchas interleukin-2 and interleukin-12; interferons such as gammainterferon; combinations such as saponin-aluminum hydroxide or QuilA-aluminum hydroxide; liposomes; iscom adjuvant; mycobacterial cell wallextract; synthetic glycopeptides such as muramyl dipeptides or otherderivatives; N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine(avridine); Lipid A; dextran sulfate; DEAE-Dextran;carboxypolymethylene, such as Carbopol® (polyacryiic polymercommercially available from B. F. Goodrich Company, Cleveland, Ohio);ethylene maleic anhydride or ethylene/malaic anhydride copolymers (EMA®,a linear ethylene/maleic anhydride copolymer having approximately equalamounts of ethylene and malaic anhydride, having an estimated averagemolecular weight of about 75,000 to 100,000, commercially available fromMonsanto Co., St. Louis, Mo.); acrylic copolymer emulsions such as acopolymer of styrene with a mixture of acrylic acid and methacrylic acidlike NeoCryl® A640 (e.g. U.S. Pat. No. 5,047,238, an uncoalesced aqueousacrylic acid copolymer of acrylic acid and methacrylic acid mixed withstyrene, commercially available from Polyvinyl Chemicals, Inc.,Wilmington, Md.); animal poxvirus proteins; subviral particle adjuvantssuch as orbivirus; cholera toxin; dimethyldioctadecylammonium bromide(DDA, commercially available from Kodak, Rochester, N.Y.); or mixturesthereof. A preferred adjuvant comprises ethylene/maleic anhydridecopolymer, copolymer of styrene with a mixture of acrylic acid andmethacrylic acid, mineral oil emulsion or combinations thereof.

To illustrate examples of how to prepare the FCV-DD1 antigen and makekilled FCV-DD1 vaccines, the virus was used to infect confluent CRFKceils at an MOI of 0.01 (typically ranges from about 0.001 to about 1.0)in tissue culture roller bottles. The virus fluids were harvested when90-100% CPE was observed. The harvested fluids were inactivated with0.04% formalin at 36° C. for 4 days. Residual formalin was neutralizedby the addition of sodium bisulfite. Killed FCV-DD1 vaccines containingapproximately 0.5 % w/v to approximately 10% w/v of FCV-DD1 were thenformulated to contain formalin-inactivated FCV-DD1 alone; FCV-DD1 incombination with killed FeLV (in the amount of approximately 5.0 % w/vto approximately 50 % w/v), FPV (in the amount of approximately 0.5 %w/v to approximately 10% w/v), FCV-255 (in the amount of approximately0.5% w/v to approximately 10% w/v), FVR (in the amount of approximately1.0% w/v to approximately 20% w/v) and C. fells (in the amount ofapproximately 0.5% w/v to approximately 10% w/v); and FCV-DD1 incombination with FPV (in the amount of approximately 0.5% w/v toapproximately 10% w/v), FCV-255 (in the amount of approximately 0.5% w/vto approximately 10% w/v) and FVR (in the amount of approximately 1.0%w/v to approximately 20% w/v). The vaccines were suitably adjuvanted;and Eagle's minimum essential media were added as the blending diluent.The amount of each antigen in the vaccines was determined using anantigen specific ELISA potency test. The vaccines were found to induceprotective immunity against hemorrhagic FCV in standard vaccinationchallenge tests. The lack of interference of other vaccine fractionsfrom the FCV-DD1 was confirmed by either challenge or serological tests.Another vaccine formulation containing FCV-DD1, FPV, FCV-255, FVR and C.felis was also prepared.

Another embodiment of the present invention involves a new method ofprotecting a feline against infection or preventing disease caused byfeline calicivirus that comprises administering to the feline animmunologically effective amount of the vaccines described herein thatcontain the isolated and purified hemorrhagic feline calicivirusFCV-DD1. Additional methods protect the feline against infection orprevent disease caused by other pathogenic agents using one or moreantigens in conjunction with FCV-DD1 such as, for example, felineleukemia virus, feline panleukopenia virus, feline rhinotracheitisvirus, feline immunodeficiency virus, rabies virus, feline infectiousperitonitis virus, Bartonella, etc. and, more preferably, a combinationof the antigens encompassing one or more non-hemorrhagic felinecalicivirus such as FCV-255, feline rhinotracheitis virus and felinepanleukopenia virus, with the optional addition, of feline leukemiavirus and/or C. felis, or other hemorrhagic FCV strains, comprising theadministration to the feline of an immunologically effective amount ofthe multivalent vaccines described herein.

A further embodiment of the present invention is drawn to the antibodiesthat are raised or produced against the FCV-DD1 antigen. The antibodiesmay be raised or produced either by in vitro or in vivo methods that arewell known to those of ordinary skill in the art. For example, a typicalin vivo method to stimulate the formation of antibodies against FCV-DD1comprises directly administering to the feline an immunologicallyeffective amount of FCV-DD1 or an antigenic subunit thereof that will besufficient to induce detectable virus-neutralizing antibody titers. Bothmonoclonal antibodies specific for the FCV-DD1 antigen and polyclonalantibodies useful to recognize different epitopes of the hemorrhagiccalicivirus strains closely related to the FCV-DD1 antigen may be usedin the practice of this invention. Further methods of this invention arebased on antigen-antibody interaction and the ability of the FCV-DD1antigen and anti-FCV-DD1 antibodies to form a detectable immune complex.Such methods include a method of defecting or diagnosing a hemorrhagicfeline calicivirus infection in a susceptible host which comprisesanalysing a biological specimen from the host and detecting the presenceof FCV-DD1 or an antibody raised or produced against FCV-DD1 in thebiological specimen and a method of detecting the anti-FCV-DD1 antibodyin a biological sample which comprises contacting the biological samplewith an antigen comprising FCV-DD1 and detecting or observing theformation of an antigen-antibody immune complex. The antigen used inthese methods is, for example, the whole virus FCV-DD1, an antigenicsub-unit of FCV-DD1 such as the immunogenic capsid protein and the like.

An additional method of the present invention to detect a carrier of thehemorrhagic feline calicivirus is warranted because the virus is highlycontagious and virulent. The infections FCV can be carried ortransmitted by an asymptomatic cat to other cats, or caretakers in ahospital setting can easily spread the infection from sick cats sheddingthe virus to healthy cats. Therefore, a method of detecting the carrierof the hemorrhagic feline calicivirus is presented that involves thesteps of (a) obtaining a test sample from asymptomatic cats (urine,serum, sputum, feces, etc.), caretakers or pet owners (eat hairs fromclothes, hands, furniture, etc.), cat cages and the like; (b) Incubatingthe test sample with, an antibody specific to FCV-DD1; (c) allowing theformation of an antibody-antigen complex; and (d) detecting the presenceof the antibody-antigen complex. This invention, further contemplatesother comparable methods making use of the specific antigen-antibodyinteraction that will be apparent to those of ordinary skill in the art.

The antigen-antibody immune complex may be detected by any standardimmunoassay that includes, but is not limited to, enzyme-linked,immunosorbent assay (ELISA), Western Blot, immunohistochemistry, flowcytometry and the like. Well-known flow cytometry techniques, forinstance, can use a device such as a Becton-Dickinson FACScan FlowCytometer that detects and measures the amount of fluorescent dye onparticles. A sample cell or specimen is labeled with afluorochrome-labeled antibody, excess unbound antibody is washed off,and then the sample is analyzed by the flow cytometer. The degree offluorescence and laser-scatter indices are observed and recorded for thesample cells passing through the cytometer. In this fashion, thedisplayed data in the form of color histograms showing the relationshipbetween the fluorochrome and light scatter characteristic confirms thepresence of bound FCV-DD1 antigen in the sample.

Other standard in vitro immunological assays for detection of viralspecific antibodies in serum or other test samples may be used throughdirect or indirect immunofluorescent methods of antibody detection andtiter determination. Indirect immunofluorescent assays may be used toscreen and identify FCV in a sample specimen. For example, a test sampleis incubated with FCV-DD1 antigen, a fragment of the major capsidprotein unique to FCV-DD1 in which the fragment can be a syntheticpeptide or a short peptide chain expressed using recombinant DNAtechniques, related hemorrhagic calicivirus isolates and the like, thencoated and stabilized on a glass slide. If anti-FCV-DD1 antibodies arepresent in the sample, a stable antigen-antibody immune complex forms.The bound antibody is then reacted with a fluroescein-conjugatedreactant and the complex is observed with a fluorescence microscope. Abrightly colored fluorescence at the antigen site confirms the positiveantibody reaction. Other standard ELISA or immunochromatographytechniques may be employed for diagnostic purposes in the detection ofantibodies or antigens coupled to an easily-assayed enzyme such as,example, detection of the presence of FCV-DD1 antigens that arerecognised by a monoclonal antibody or test for antibodies thatrecognise the FCV-DD1 antigen, ELISA, in particular, can supply a usefulmeasurement of either antigen or antibody concentrations. Alternatively,the FCV-DD1 antigen may be attached to a solid support such as apolystyrene surface of a microwell test strip. The test sample such ascat serum is washed to remove residual serum and thenperoxidase-conjugated enzyme is added. A detectable substrate such asthe colorless tetramethylbenzidine/hydrogen peroxide is also added andhydrolyzed by the enzyme. The chromogen changes to a blue color. Afterthe reaction is stopped with the addition of acid, the colorlesstetramethylbenzidine/hydrogen peroxide changes to yellow. In the finalanalysis, the intensity of the color detects the presence of theantibody-antigen complex in the sample.

The new FCV strain has been deposited under the conditions mandated by37 C.F.R. §1.308 and maintained pursuant to the Budapest Treaty in theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A. Specifically, the FCV-DD1 sample hasbeen deposited in the ATCC on Sep. 9, 2004 and has been assigned ATCCPatent Deposit Designation PTA-6204.

The following examples demonstrate certain aspects of the presentinvention. However, it is to be understood that these examples are forillustration only and do not purport to be wholly definitive as toconditions and scope of this invention. It should be appreciated thatall scientific and technological terms used herein have the same meaningas commonly understood by those of ordinary skill in the veterinary andpharmaceutical arts. For purposes of this invention, any reference inthe specification or the claims to the FCV-DD1 strain includes the viralclones derived from the FCV-DD1 isolate. These viral clones may bereadily substituted for FCV-DD1 in all aspects of the vaccines, methods,etc. described herein. It should be further appreciated that whentypical reaction conditions (e.g., temperature, reaction times, etc.)have been given, the conditions bosh above and below the specifiedranges can also be used, though generally less conveniently. Theexamples are conducted at room temperature (about 23° C. to about 28°C.) and at atmospheric pressure. All parts and percents referred toherein are on a weight basis and all temperatures are expressed indegrees centigrade unless specified otherwise.

A further understanding of the invention may be obtained from thenon-limiting examples that follow below.

EXAMPLE 1 Failed Attempt to Isolate Hemorrhagic Feline Calicivirus

Two 25-cm² tissue culture flasks of FCV-ari (labeled 1:100 and 1:1000)were obtained from Dr. Neils Pederson, School of Veterinary Medicine, UCDavis (N. C. Pederson et. al., “An isolated epizootic ofhemorrhagic-like fever in cats caused by a novel and highly virulentstrain of feline calicivirus,” Veterinary Microbiol. 73:281-300 (May2000)). The FCV-Ari flask labeled 1:1000 was frozen and thawed. Then 1mL of the culture fluid was used to infect one 850-cm² tissue cultureroller bottle of confluent Crandell Feline Kidney Cells (CRFK) (R. A.Crandell et al., “Development, characterization, and viralsusceptibility of a feline (Felis catus) renal cell line (CRFK),” InVitro 9:176-185 (1973)). Sixteen hours later, the roller bottle wasfrozen, thawed, and aliquoted as a working stock.

The initial FCV-Ari “working stock” was used to inoculate cats in orderto confirm that the material contained hemorrhagic calicivirus. The catsinoculated with the FCV-Ari “working stock” elicited extreme clinicalsigns such as high pyrexia, edema, dehydration, and death. Thus, theFCV-Ari “working stock” was confirmed to contain the hemorrhagiccalicivirus.

The FCV-Ari “working stock” was diluted to a titer of approximately 10⁵TCID₅₀ per mL and 0.2 μm filtered. The filtered FCV-Ari was used forpurification/isolation of the most virulent calicivirus strain. Thefiltered FCV-Ari was serially diluted and used to infect 24-well tissueculture plates confluent with CRFK cells. The well of the highestdilution which displayed a cytopathic effect (CPE) on the CRFK cells washarvested, frozen, quick-thawed, serially diluted, and used to infectanother 24-well tissue culture plate confluent with CRFK cells. Thisprocedure was repeated two times for a total of three rounds ofpurification and isolation.

The purified FCV-Ari was formalin inactivated and used to blend akilled, monovalent vaccine. This FCV-Ari (failed) vaccine was put intocats to measure the serological response to vaccination. The vaccine didnot induce virus-neutralizing antibody titers although antibodiesagainst the virus were induced as confirmed by ELISA.

Specifically, the study with the purified, killed FCV-Ari vaccine used20 cats, five cats per group in doses of 0.5 % v/v, 2% v/v and 8% v/vwith five controls (no injections). Each of the fifteen cats received2×1 mL doses subcutaneously at the nape of the neck three weeks apart.At one and two weeks following the final vaccination, there were nomeasurable serum neutralization (SN) titers to FCV at a time when FCV SNtiters are typically at their peak.

To re-test and confirm initial findings, four more cats received 2×1 mLdoses three weeks apart with the purified, killed FCV-Ari vaccine. Oneweek following the final vaccination, there were no measurable serumneutralization antibody titers (all <2) at the timepoint when serumneutralization titers for FCV are typically the highest.

The purified FCV-Ari (live) was also used to inoculate two groups ofcats. The two groups of cats exhibited no clinical signs characteristicof a hemorrhagic calicivirus infection such as high temperatures, edema,pyoderma, alopecia, etc. Therefore, it was confirmed that, the strain,purified from the “working stock” was not the hemorrhagic calicivirusstrain. Because the vaccine also did not induce neutralizing antibodies,further work and development of this strain ceased.

EXAMPLE 2 Isolation of FCV-DD1 by Limiting Dilution Cloning

The original FCV-Ari received from Dr. Pedersen, labeled 1:100, was usedfor three rounds of limiting dilution cloning to purify and isolateFCV-DD 1. The FCV-Ari 1:100 sample was incubated with the antiseragenerated from the original FCV-Ari (killed) vaccination to neutralizethe FCV strain isolated from the first purification/isolation ofFCV-Ari. The virus was serially diluted and used to infect 24-welltissue culture plates confluent with CRFK cells and the well of thehighest dilution which showed a cytopathic effect (CPE) on the CRFKcells was harvested, frozen, and quick thawed. The harvested clones wereevaluated by serum neutralization assays. (For a general description ofthe limiting dilution cloning method and serum neutralization assaysused for distinguishing and isolating FCV strains, see H. Poulet et al.,“Comparison between acute oral/respiratory and chronicstomatitis/gingivitis isolates of feline calicivirus: pathogenicity,antigenic profile and cross-neutralisation studies,” Arch. Virol.145:243-261 (2000).) Each viral clone of FCV-Ari was incubated with feeantisera generated from the first purification killed vaccine, or withthe antisera generated from challenge with the live “working stock.” Theresults from this serum, neurotization assay showed that there was morethan one strain of calicivirus in the original sample. The viral clonesthat were not selected and discarded were, those that were neutralizedby the antisera specific to the undesired product of the firstpurification. The virus clones selected for the subsequent rounds ofpurification were those that were neutralized by antisera to theoriginal virus sample from Dr. Pedersen yet were not neutralized byantisera specific to the undesired product of the first purification.This pattern of virus clone selection, harvesting the highest dilutioncontaining CPE, was repeated for a total of three rounds. The resultingclone, designated FCV-DD1, was chosen for biological studies.

Specifically, the FCV-Ari sample was neutralized and repurified throughlimiting dilution cloning. To neutralize the FCV-Ari virus, the originaltissue culture of FCV-Ari sample was diluted to 1:200 in 1×MEM (modifiedEagle's medium). The antisera generated from the original FCV-Ari(killed) vaccination (Vaccinate α-Ari serum) was diluted 1:50 in 1×MEM.To 2 mL of diluted anti-Ari serum was added 2 mL of diluted virus. Thevirus/antisera mixture was incubated for about 1 hour at 37° C.

From a pilot FCV-Ari purification using neutralized virus, it was foundthat the CPE in 24-well plates was positive up to 10⁻² dilution (10⁻²wells were 50% CPE and 10⁻³ were 0% CPE). Based on this information, thevirus was diluted to achieve CPE in about 50% of the wells of CRFKcells. Three dilutions were done, over the goal by fourfold, at the goaland under the goal by fourfold.

One 24-well plate was used for each dilution. All 24 wells were used asreplicates of the same dilution. These plates were incubated at 37° C.with 5% CO₂ for 4 days. Wells which were positive for CPE in thedilution that gave less than or approximately equal to 50% CPE in the 24replicates (i.e., ≦12 positive wells) were harvested.

For Round @1 of the limiting dilution cloning procedure, crossneutralization analysis was performed on the harvested clones. Eachharvest was diluted 1:200 and 1:1000 in 1×MEM and mixed with dilutionsof either Challenge α-Ari (antiserum generated from challenge with thelive “working stock”) or Vaccinate α-Ari (antiserum from the firstpurification killed vaccine), in replicates of two. The virus-serummixture was incubated at 37° C. for 1 hour and then plated onto CRFKceils in 96-well plates. The plates were incubated for 3 days and readfor CPE. Results from the first round of limiting dilution cloning andcross neutralization screening are shown in the below Table 1.

Five, harvested clones (AB2, BC1, BC4, CB4 and DD1) were neutralised byantisera to the original virus sample from Dr. Pedersen yet were notneutralized by antisera specific to the undesired product of the firstpurification. They were selected for Round @2 of purification.

For Round @2 of the limiting dilution cloning procedure, the CPE in24-well plates was found to be positive up to 1.0⁻⁵ to 10⁻⁶. Threedilutions were done, over the goal, by fourfold, at the goal and underthe goal by fourfold. One 24-well plate was used for each dilution. All24 wells were used as replicates of the same dilution. These plates wereincubated at 37° C. with 5% CO₂ for 4 days. Wells that were positive forCPE in the dilution that gave ≦50% CPE in the 24 replicates wereharvested.

The above steps were repeated for the third round of limiting dilutioncloning.

TABLE 1 FCV-Ari Limiting Dilution Cross Neutralization Screening HarvestVirus Challenge Vaccinate ID Dilution α-Ari α-Ari AB2 1:200 >256 <2 AB21:1000 >256 <2 BC1 1:200 >256 <2 BC1 1:1000 >256 <2 BC4 1:200 >256 <2BC4 1:1000 >256 <2 CA4 1:200 >256 <2 CA4 1:1000 >256 <2 CB2 1:200 >256<2 CB2 1:1000 >256 <2 CB3 1:200 >256 <2 CB3 1:1000 >256 <2 CB41:200 >256 <2 CB4 1:1000 >256 <2 CC5 1:200 >256 <2 CC5 1:1000 >256 <2CD1 1:200 >256 37 CD1 1:1000 >256 <2 DA5 1:200 >256 <2 DA5 1:1000 >256<2 DB1 1:200 >256 <2 DB1 1:1000 >256 <2 DB5 1:200 >256 <2 DB51:1000 >256 <2 DC2 1:200 >256 <2 DC2 1:1000 >256 <2 DD1 1:200 >256 <2DD1 1:1000 >256 <2 DD2 1:200 >256 <2 DD2 1:1000 >256 <2 DD4 1:200 >256<2 DD4 1:1000 >256 <2

EXAMPLE 3 Isolation of FCV-DD1 Strain

The one harvested clone, DD1, was selected from round @3 and used toinfect 850 cm² tissue culture roller bottle of confluent CRFK cells atMOI (Multiplicity of Infection) of approximately 0.003. The virus fluidwas harvested from the roller bottle when 100% CPE was observed, frozenat −50° C. for 4 hours and then quick thawed in 37° C. water bath. Thevirus fluid was centrifuged in a Beckman GS-6R Centrifuge (commerciallyavailable from Beckman instruments, Inc., Fullerton, Calif.) at 3000 rpmfor 20 minutes, and the cell-free supernatant was aliquoted into 81×1 mLsample vials and stored at −80° C.

EXAMPLE 4 Physilogical Challenge Studies

The isolated and purified FCV-DD1 strain prepared in Example 3 wasinoculated into cats. One challenge group of three cats were usedwherein each cat received 6.3 logs of virulent FCV-DD1 by administrationof 0.25 mL per nostril and 0.5 mL orally for a total of 1 mL. The catsexhibited typical hemorrhagic calicivirus clinical signs. Extremely hightemperatures appeared in all 3 cats after 1 day. The edema (swelling)started on the fifth observation day. Ulcerations, both external andoral, appeared on the sixth observation day. Two-thirds of the cats wereeuthanized (exanguinated) on the sixth observation day since they hadbecome moribund. The third cat was moribund with failing temperatures onthe seventh observation day and the study was ended. The results fromthis challenge study prove that the strain of calicivirus purified andisolated from the original FCV-Ari sample, designated FCV-DD1, was atrue hemorrhagic feline calicivirus strain.

In the foregoing, there has been provided a detailed description ofparticular embodiments of the present invention for the purpose ofillustration and not limitation. It is to be understood that all othermodifications, ramifications and equivalents obvious to those havingskill in the art based on this disclosure are intended to be includedwithin the scope of the invention as claimed.

1-40. (canceled)
 41. An isolated antibody that specifically binds to thehemorrhagic feline calicivirus FCV-DD1, said virus having ATCC PatentDeposit Designation PTA-6204, and does not bind to non-hemorrhagicfeline calicivirus strains, wherein said antibody specifically binds toa fragment of the major capsid protein unique to said FCV-DD1.
 42. Anantibody of claim 41, wherein said antibody is a monoclonal antibody.43. A method of stimulating the formation of an antibody of claim 41 inan animal capable of making antibodies, comprising administering to saidanimal an immunologically effective amount of a hemorrhagic felinecalicivirus FCV-DD1, ATCC Patent Deposit Designation PTA-6204, or anantigenic subunit thereof.
 44. A method of claim 45, wherein said animalis a feline.
 45. A method of claim 46, wherein said animal is a cat. 46.A method of claim 45, wherein an attenuated form of hemorrhagic felinecalicivirus FCV-DD1 is administered.
 47. A method of claim 48, whereinsaid animal is a feline.
 48. A method of claim 49, wherein said animalis a cat.
 49. A method of determining whether a susceptible host is acarrier of hemorrhagic feline calicivirus, comprising: (a) contactingthe biological sample from said susceptible host with an antibody ofclaim 41 under conditions effective to induce the formation of anantibody-antigen complex between an antigen specific to said hemorrhagicfeline calicivirus in said sample and said antibody; and (b) detectingthe presence or absence of the antibody-antigen complex, wherein thepresence of the antibody-antigen complex indicates that the host is acarrier of hemorrhagic feline calicivirus.
 50. A method of claim 51,wherein said host is a feline.
 51. A method of claim 52, wherein saidfeline is a cat.
 52. A method of detecting or diagnosing a hemorrhagicfeline calicivirus infection in a susceptible host, which comprisesanalyzing a biological sample from the host and detecting the presenceof an antibody of claim 41 in the biological specimen.
 53. A method ofdetecting an antibody of claim 41 in a biological sample, comprising (a)contacting the biological sample with an antigen comprising an antigenicdeterminant specific to feline hemorrhagic calicivirus FCV-DD1,deposited as ATCC PTA-6204, under conditions effective to induce theformation of an antibody-antigen complex between an antibody in saidsample that specifically binds to said hemorrhagic feline calicivirusand said antigen; and (b) detecting the presence or absence of theantibody-antigen complex, wherein the presence of the antibody-antigencomplex indicates that the sample contains said antibody.
 54. A methodof claim 55, wherein the antigen is an antigenic subunit of FCV-DD1,deposited as ATCC PTA-6204.
 55. A method of claim 55, wherein theantibody-antigen complex is detected by an enzyme-linked immunosorbentassay (ELISA).
 56. A method of claim 55, wherein the antibody-antigencomplex is detected by Western blot.
 57. A method of claim 55, whereinthe antigen-antibody immune complex is detected by flow cytometry.
 58. Amethod of claim 55, wherein the antibody-antigen immune complex isdetected by immunofluorescent assay (IFA).
 59. An enzyme-linkedimmunosorbent assay (ELISA) for detecting an antibody of claim 41, whichcomprises a solid support coated with an antigenic determinant specificto feline calicivirus FCV-DD1, deposited as ATCC PTA-6204, and adetectable substrate that indicates when the antibody binds to theantigen.